The genus Rachicladosporium: introducing new species from sooty mould communities and excluding cold adapted species

The fungal genus Rachicladosporium (Cladosporiales, Cladosporiaceae), typified by cladosporium-like Rachicladosporium luculiae, includes a morphologically diverse assemblage of species. The species of this genus were reported from different substrates, habitats and environments, including plant leaves and needles, twig, black mould on baobab trees, rocks and insects. In this study, four new Rachicladosporium species (R. europaeum, R. ignacyi, R. kajetanii, R. silesianum) isolated from sooty mould communities covering leaves and needles of trees and shrubs in Poland are described. The new species are delineated based on morphological characteristics and molecular phylogenetic analyses using concatenated ITS, LSU, and rpb2 sequences. All newly described species are nested in the main Rachicladosporium lineage (centred around the type species), which contains species that are able to grow at 25 °C. By contrast, four cold adapted, endolithic species known from Antarctica (R. antarcticum, R. aridum, R. mcmurdoi) and Italian Alps (R. monterosanum) form distant phylogenetic lineage and do not grow at this temperature. Therefore, they are accommodated in the new genus Cryoendolithus, typified by Cryoendolithus mcmurdoi.


Phylogenetic analyses
Megablast searches in GenBank 39 were used to find closest hits for the obtained sequences.Subsequently, newly generated sequences and reference sequences of representatives of the Cladosporiales, including all described Rachicladosporium species, were used to assemble a concatenated ITS, LSU, rpb2 alignment and conduct phylogenetic analyses.Pseudocercospora eucalyptorum was used as an outgroup (Table 4).
The ITS, LSU and rpb2 datasets were independently aligned using the MAFFT algorithm 40 in Geneious Prime R11.The final phylogenetic tree was constructed using the concatenated alignment of these three genes.Phylogenetic analyses were carried out using RAxML-NG v. 1.1.1 and MrBayes v. 3.2.6 41,42 , respectively.For maximum likelihood (ML) and Bayesian inference (BI) analyses the best substitution models were selected using ModelTest-NG v. 0.2.0 43 .Maximum likelihood analysis was conducted with bootstrapping 1000 replicates.Bayesian inference was performed with the following parameter: 1 000 000 generations with trees sampled every 100th generation.The first 25% of trees were discarded as burn-in.Maximum likelihood bootstrap (MLB) support values above 70% and Bayesian posterior probabilities (BPP) above 0.95 were considered strongly supported.FigTree v1.4.3 was used for visualising the final phylogenetic tree.

Results
In total, eight strains assigned to the genus Rachicladosporium were obtained from sooty mould communities in southern Poland.Amongst the analysed trees and shrubs, the Rachicladosporium strains were found only on leaves or needles of five species: Betula pendula (one strain), Pinus nigra (three strains), Rosa sp.(one strain), Symphoricarpos albus (two strains) and Tilia cordata (one strain).Morphological characteristics of isolated fungi are given in species descriptions.All tested strains grew in all analysed temperatures (6 °C, 15 °C and 25 °C) on both culture media (MEA and PDA), with the best growth rate observed at 15 °C (Table 2).The literature data of thermal preferences for previously described Rachicladosporium species, whenever available, showed that most of them grew at 25 °C, except R. antarcticum, R. mcmurdoi and R. monterosanum for which the growth at this temperature was not observed (Table 3).

Phylogenetic analyses
Megablast searches in GenBank 39 revealed similarities of the sequences of the studied strains to sequences of the Rachicladosporium species.The concatenated ITS, LSU, rpb2 alignment contained sequences belonging to 32 species, including 31 species of the Cladosporiales and a member of the Mycosphaerellales (Pseudocercospora eucalyptorum) used as an outgroup.The alignment contained a length of 1744 characters (with gaps), including 678 bp for the rDNA ITS, 759 bp for the rDNA LSU and 307 bp for the rpb2.The best matching substitution models selected for single locus alignments in the ML analysis were as follows: TIM1ef + I + G4 for both ITS and LSU, and TrNef + G4, JC and TPM2uf + G4 for rpb2 (three codons).The BI analysis was performed with the following substitution models: SYM + I + G4 for both ITS and LSU, and K80 + G4, JC and HKY + G4 for rpb2 codons.
ML and BI analyses resulted in similar tree topologies.The best scoring maximum likelihood phylogenetic tree is shown on Fig. 1.Rachicladosporium species were split into three groups: (i) main lineage containing type species, Rachicladosporium luculiae, (ii) single species lineage containing Rachicladosporium iridis, (iii) psychrophilic lineage containing three Antarctic and one Italian Alpine species.The main Rachicladosporium lineage was fully supported in both ML and BI analyses.Rachicladosporium iridis was strongly (MLB = 98%) or fully supported (BPP = 1) as a sister species to the main Rachicladosporium lineage.A clade grouping psychrophilic species (MBL = 100%, BPP = 1), formed a strongly supported (MLB = 88%) or not supported (BI analysis) sister lineage to the remaining two lineages.
The sooty mould strains formed four independent subclades in the main Rachicladosporium lineage.Sequences from cultures F95, G21, G24, G398 (described below as R. ignacyi) were almost identical in ITS (F95 differed in one bp from remaining strains) and identical in LSU and rpb2 and formed a fully supported clade (MLB = 100%, BPP = 1) that was sister (though not supported) to Rachicladosporium pini.Sequences from cultures G232 and G395 (described below as R. silesianum) were identical in all analysed loci and formed a fully supported clade (MLB = 100%, BPP = 1) that was sister (and strongly supported, MLB = 85%, BPP = 0.95) to Rachicladosporium cboliae.Sequences from culture G42 (described below as R. kajetanii) were sister (though not supported) to Rachicladosporium alpinum and R. paucitum.Sequences from culture G231 (described below as R. europaeum) were sister (and fully supported, MLB = 100%, BPP = 1) to Rachicladosporium inconspicuum.Sequences of R. europaeum and R. inconspicuum differed in two base pairs (bp) in ITS, six bp in LSU and 39 bp in rpb2.www.nature.com/scientificreports/

Discussion
This study reports on so far unknown Rachicladosporium species isolated from sooty mould communities on the surface of the leaves or needles of woody plants in Poland.The habitat occupied by sooty moulds is an extreme environment, rich in sugars and poor in water 32 , from which none Rachicladosporium species has been recorded so far.Rachicladosporium africanum was described from black mould on the bark of baobab (Adansonia digitata) trees in Africa 10 .Indeed, fungi involved in the black mould on baobab trees were commonly considered to be "sooty moulds" but are different in that they penetrate infected tissues and cause host reaction 10 .Eight isolated Rachicladosporium strains, obtained amongst hundreds of fungal strains isolated from sooty moulds, represented four species that were previously undescribed.Phylogenetically, they reside in the main Rachicladosporium lineage containing type species, Rachicladosporium luculiae.The morphology is complex in Rachicladosporium ignacyi, but simplified in the remaining newly described species that contain only hyphae, chlamydospores and arthroconidia.Our hypothesis that they are distinct species was supported by molecular data.According to the phylogenetic analyses they form independent subclades scattered over the subclades of Rachicladosporium species with completely different morphologies (Fig. 1; see notes in the species descriptions).Although described species are probably not host-specific, most strains were obtained from Pinus nigra and the types of Rachicladosporium ignacyi and R. silesianum are from this host plant.Another species, Rachicladosporium pini, has been previously described from needles of Pinus monophylla 9 .Unclassified species were also reported as endophytes of Pinus densiflora, P. koraiensis, P. rigida, P. thunbergii and P. wallichiana 21,23 .This indicates that Pinus may be the favourite genus for Rachicladosporium and harbour further undescribed species.
The simple morphology in the three Rachicladosporium species described here from sooty mould communities is morphologically convergent with, for example, rock inhabiting fungi, which often produce similar morphological structures 14,44,45  ultraviolet radiation, temperature fluctuations and limited amount of nutrients 46 , and probably the development of similar simple morphologies is an adaptation to occurrence in similar extreme environments.In fact, some Rachicladosporium species were described from rocks 14,16 .These include R. alpinum, R. inconspicuum and R. paucitum from the main Rachicladosporium lineage and R. antarcticum, R. aridum, R. mcmurdoi and R. monterosanum from the psychrophilic lineage.Interestingly, R. europaeum and R. kajetanii described here from sooty mould communities are closely related to R. inconspicuum and R. alpinum/R.paucitum, respectively.Close relationships between sooty mould and rock inhabiting fungi were already reported for the orders Capnodiales (s.l.) and Chaetothyriales 32,47 .It is considered that rock inhabiting fungi evolved from sooty moulds.Honeydew dropped from the plants into the rocks may have been the vehicle in their evolution 32 .This is, however, still a poorly known phenomenon deserving in-depth analyses.
The main Rachicladosporium lineage contains species that are able to grow at 25 °C.This refers also to rock inhabiting fungi, isolated from the Alps, nested within this lineage.By contrast, four cold adapted, rock inhabiting www.nature.com/scientificreports/species from Antarctica (three species) and Italian Alps (one species) that form psychrophilic lineage, do not grow at this temperature (Table 3) 14,16 .The phylogenetic distance of this lineage is comparable between genetic distances in related genera of the Cladosporiales.Therefore, the reassessment of published morphological characters and thermal preferences 14,16 together with molecular phylogenetic analyses conducted in this study give arguments to include them in a distinct genus, named here as Cryoendolithus.Rachicladosporium iridis may belong to yet another undescribed genus to be separated from the Rachicladosporium s. str.Since it forms a single species lineage, the eventual reclassification into separate genus needs more species and sequences.In summary, the results of this work expand the knowledge on the diversity and ecological preferences of the genus Rachicladosporium.It also excludes from the genus Rachicladosporium cold adapted species and classifies them in the distinct genus Cryoendolithus.It is likely that further species will be described in this genus as this may be deduced from the reports of unclassified species available in the literature.

Figure 1 .
Figure 1.Phylogenetic tree of selected members of the Cladosporiales, including all sequenced Rachicladosporium species, obtained from a maximum likelihood analysis of the combined multi-locus alignment (ITS, LSU, rpb2).The positions of new Rachicladosporium species and new genus Cryoendolithus are indicated in bold.Ex-type cultures are indicated with superscript T. Numbers above branches indicate maximum likelihood bootstrap (MLB) support values > 70% and Bayesian posterior probabilities (BPP) > 0.9, respectively (MLB/BPP).Pseudocercospora eucalyptorum was used as an outgroup.The scale bar represents the expected number of changes per site.

Table 3 .
Thermal preferences of previously reported Rachicladosporium species.R. = Rachicladosporium.CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CCFEE: Culture collection of fungi from extreme environments, Tuscia University, Viterbo, Italy; CMW: Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; CPC: Culture collection of Pedro Crous, housed at the Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; MUT: Mycotheca Universitatis Taurinensis, Department of Plant Biology of the University of Turin, Turin, Italy.

Table 4 .
List of species, with country of origin, host/substrate, strain and GenBank accession numbers, used in phylogenetic analyses.Superscript T denotes ex type strain (epitype, holotype or neotype).CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CCFEE: Culture collection of fungi from extreme environments, Tuscia University, Viterbo, Italy; CMW: Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; CPC: Culture collection of Pedro Crous, housed at the Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; MUT: Mycotheca Universitatis Taurinensis, Department of Plant Biology of the University of Turin, Turin, Italy.-Indicates unavailable sequence.